Protein synthesis is a multi-step process that begins with transcription, in which the genetic information encoded by DNA is transferred to an mRNA template. Transcription starts with the process of initiation. RNA polymerase binds to a specific region of DNA, called the promoter, which is usually located upstream of the gene to be transcribed. Many promoters, but by no means all, contain a common (conserved) sequence, called a consensus sequence.
In prokaryotes, the promoter consists of two short sequences at positions −10 and −35 upstream of the transcription start site. The sequence at −10 usually consists of the six nucleotides “TATAAT” (TATA box). The other sequence at −35 usually consists of the six nucleotides TTGACA. Most promoters differ from one another in the actual base sequence and distance from the transcription start site of the consensus sequence. This variability is believed to lead to different frequencies with which promoters initiate the transcription, that is, promoter strengths. The promoter is thus one of the important factors that determines the efficiency of protein production, and extensive research is being conducted to develop strong and specific promoters in various microorganisms.
Pichia pastoris, a species of methylotrophic yeast, is emerging as an important bio-model useful in the study of methanol metabolism and peroxisome production, and as a protein production host system superior to the conventional yeast, Saccharomyces cerevisiae. In addition, Pichia pastoris is recognized as a useful industrial resource that is useful in environmentally-friendly bio-processes thanks to its characteristic metabolism and physiological activity.
Pichia pastoris oxidizes methanol through formaldehyde and formate to carbon dioxide. These reactions are catalyzed by alcohol oxidase (AOX), formaldehyde dehydrogenase (FLD) and formate dehydrogenase (FMDH), respectively. Pichia pastoris has two alcohol oxidase genes (AOX1 and AOX2), which have respective inducible promoters. Thanks to this high activity, the AOX1 promoter is usually used to express a recombinant protein in yeast, while the AOX2 promoter (U.S. Pat. No. 5,032,516) is known to exhibit relatively weak activity (J. Tschopp et al. Nucleic Acids Res. 1987, vol. 15, pp. 3859-3876). In addition, an FLD gene promoter has been developed (D. Resina et al. Journal of Biotechnology, 2004, vol. 109, pp. 103-113). A glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter, known as a strong constitutive promoter in various microorganisms, has also been developed for Pichia pastoris (Waterham et al. Gene, 1997, vol. 186, pp. 37-44).
Most frequently used in the production of recombinant proteins through Pichia pastoris are the promoters which are involved in methanol metabolism and are strongly induced by methanol. When using a promoter of a gene involved in methanol metabolism, care must be taken to avoid fire, because methanol, a highly combustible fuel, is required for transcription. In this case, the factory facility must include special instruments and is required to be carefully inspected. In addition, it is difficult to control the efficiency of protein expression with the GAP promoter. When having negative effects on the growth of the cell, other constitutive promoters are also difficult to use in the mass production of proteins because they cause cells to be instable.
Therefore, there is a need for a strong promoter that is inducible enough to regulate the expression of recombinant proteins at a desired time point suitable for the mass production or overexpression of proteins, and that requires for the induction thereof either an inducer that is not affected by the process, or no inducer at all.